Natural products (NPs) derived from microbes, plants, and animals have proven to be among the richest sources of bioactive molecules for use as therapeutics including most antibiotics. Even today, with the rate of novel NP discovery dramatically slowed, NPs form the basis of approximately one third of the top 200 drugs currently sold worldwide and approximately 45% of the new chemical entities approved as drugs over the past 25 years (McGrath et al. 2010. J. Chem. Educ. 87:1348-1349).
Compared to small molecules derived from synthetic and combinatorial chemistry methods, NPs are generally much larger (higher molecular weight), contain more complex chemistry (multiple chiral centers), and provide numerous hydrophilic and hydrophobic surfaces to mediate multiple protein interactions (Guiguemde et al. 2012. Chem. Biol. 19:116-129).
When combined with evolutionary selection for biological activity, these characteristics explain why NPs have proven to be such a rich source of therapeutic molecules. Not only do NPs occupy more complex chemical space than molecules generated by synthetic or combinatorial chemistry, but NPs are often difficult or currently impossible to replicate using synthetic approaches. Despite the proven potential of NPs, efforts to discover novel NPs both in academia and industry have languished in the past decades (Watve et al. 2001. Arch. Microbiol. 176:386-390). Most pharmaceutical companies have shut down their NP discovery programs due to cost, inefficiency, lengthy timelines, and dwindling returns. A major reason for this can be understood by examining the challenges faced by traditional NP study.
A traditional approach to NP discovery begins with the isolation and growth of a microbial organism. Fermentation extracts can then be fractionated and assayed for desired bioactivity. Continued bioassay-guided fractionation eventually results in a purified active molecule which can be further characterized and structurally analyzed. The process is untargeted and, for the most part, blind until the molecule is purified and identified. A major limitation to this approach to NP discovery is the fact that a significant portion of biosynthetic gene clusters encoded in the genomes of organisms are transcriptionally silent, and thus are not producing the encoded molecule. Despite the ability to modify many parameters in laboratory growth conditions, including media, temperature, stage of growth, etc. most of these silent biosynthetic gene clusters remain recalcitrant to activation. Thus, a significant portion of NP space and diversity is essentially going unnoticed.
A number of bacteria have attracted pharmacological and commercial interest as prolific producers of antibiotics and other secondary metabolites. Genes for antibiotics and other secondary metabolites are typically clustered in the genomes of these bacteria and metabolite production is influenced by a wide variety of environmental and physiological signals. Expression of secondary metabolism genes in bacteria is typically subject to multi-level control, which generally involves a specific activator that controls transcription of the pathway, and global control that allows tuning of gene expression in response to growth conditions (Xu et al. 2012. PloS ONE. 7(7):e41359).
Conjugation and/or protoplasting techniques for transformation and integration of genes of interest have been determined through experimentation for few Actinomycetes. The majority of Actinomycetes have not been successfully transformed using these approaches. The time needed for experimentation in modifying the protocols for each Streptomyces species tested can be extremely limiting. (Keiser et al. 2000. Practical Streptomyces Genetics. John Innes Centre).
Accordingly, there remains a need for developing compositions and methods for activating individual genes and gene clusters that are otherwise transcriptionally silent, poorly expressed, or poorly transcribed, such as when cultured in the laboratory. There is also a great need for the identification of new natural products that are of therapeutic and/or commercial use. The present disclosure meets these needs and provides related advantages as well, such as providing a transcription factor that activates a transcriptionally silent gene or gene cluster, industrializing the process in a standard fashion across a wide-range of bacterial strains (e.g., Actinomycete strains) in a high-throughput and cost-effective manner.